Research Summary Glutamate transporters harness the ionic gradients present across the cellular membranes for the concentrative uptake of glutamate. Normal function of glutamate transporters is required for the rapid removal of glutamate from the synapse to terminate synaptic transmission and prevent excitotoxicity. Altered function of glutamate transporters has been implicated in neurological diseases such as Alzheimer's and Amyotrophic lateral Sclerosis (ALS or Lou Gehrig's disease). There have been major advances in understanding the structure, function and dynamics of glutamate transporters but fundamental questions remain. Here we seek to investigate the process of the coupled binding of Na+ and substrate, which is a critical aspect of the transport mechanism. The coupled binding is proposed to involve the movement of the extracellular gate. A challenge in elucidating the coupling mechanism has been the lack of an assay for monitoring the movement of the extracellular gate. Further, investigating the Na+ binding sites is complicated due to an extensive involvement of the protein backbone in Na+ coordination. Here we overcome these challenges by using unnatural amino acid (UAA) mutagenesis. UAA mutagenesis is a powerful method for protein modification, compared to traditional mutagenesis, that allows a large variety of side chain modifications and permits the modification of the protein backbone. We carry out our investigations on GltPh, an archaeal homolog that has been extensively used to probe the structure and dynamics of glutamate transporters. We carry out UAA mutagenesis of GltPh using two approaches, in vivo nonsense suppression and protein semisynthesis. We use UAA mutagenesis to develop a fluorescence assay to monitor the movement of the extracellular gate, to modify residues in the substrate binding site and to modify the protein backbone to perturb the Na+ binding sites. Using these approaches, we probe the role of residues in the substrate binding site (Aim 1) and the role of the Na+ binding sites (Aim 2) in the coupled binding of Na+ and substrate to GltPh. The research proposed is significant as it will provide valuable information on the transport mechanism in glutamate transporters. Further, the research proposed will establish novel methodologies for monitoring protein dynamics and for membrane protein engineering using protein semisynthesis.